Grantee Research Project Results
1999 Progress Report: Meaningful Detection of Known and Emerging Pathogens in Drinking Water
EPA Grant Number: R826828Title: Meaningful Detection of Known and Emerging Pathogens in Drinking Water
Investigators: Cangelosi, Gerard A.
Institution: University of Washington , Seattle Biomedical Research Institute
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 1998 through August 31, 2001
Project Period Covered by this Report: September 1, 1998 through August 31, 1999
Project Amount: $360,609
RFA: Drinking Water (1998) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
Microbial contaminants of drinking water often are difficult to detect by laboratory cultivation, and polymerase chain reaction (PCR) detection of their genetic material can be of uncertain significance because of the detection of dead cells or their remnants. We hypothesize that PCR or DNA probe assays can differentially detect viable bacterial cells when the assays are targeted to one of two nonstandard nucleic acid analytes, pre-rRNA and BrdU-DNA. As intermediates in rRNA synthesis, pre-rRNA molecules are abundant in growing bacterial cells, but rare in nongrowing or nonviable cells. BrdU is a thymidine analog that is incorporated into DNA during DNA replication, which occurs in viable cells but not in nonviable cells. Both of these analytes can be detected in species-specific fashion and both are diagnostic of cells capable of nucleic acid synthesis and growth. We will use pre-rRNA and BrdU-DNA assays to study the starvation kinetics of Mycobacterium avium in drinking water and the resistance of this pathogen to disinfection. We also will determine whether the assays can distinguish replicative from nonreplicative forms of Helicobacter pylori in water.Progress Summary:
During Year 1, we: (1) developed quality assurance protocols for manufacturing novel reagents used in the BrdU-DNA test, (2) developed methods for measuring cellular pre-rRNA copy number relative to genome copies, (3) developed pre-rRNA and mature rRNA tests for rapidly measuring the toxic effects of chloride on M. avium in water, and (4) identified a cell-wall staining phenotype and a corresponding genetic marker, which are positively correlated with chloride resistance in M. avium.
Year 1 efforts have focused on optimization of pre-rRNA and BrdU-DNA assays and use of these assays to study the growth physiology and chloride resistance of M. avium in drinking water. The most significant results have been in assay use. Isolates of M. avium have long been known to segregate into colony morphotypes that vary with regard to drug susceptibility and virulence. We identified additional morphotypic variation detectable by growing M. avium colonies on agar plates containing Congo red (CR), a diazo dye that binds to most lipoproteins. All M. avium clinical isolates formed red colonies on CR agar, and about 40 percent of them also formed white colony variants that were resistant to multiple antibiotics and to chloride. Red-white variation was independent of previously-known morphotypic switches in M. avium. In addition to being more resistant than red variants, white variants were more flocculent and more adherent to glass and plastic surfaces, suggesting a possible role in biofilm formation. We went on to identify a genetic marker that cosegregates with the drug-resistant white phenotype. This marker, an insertion event provisionally named ORF1::IS999, is the first genetic marker of multiple-antibiotic and disinfectant resistance to be identified in M. avium. With regard to drinking water quality, we found that red and white variants were equally stable in water at room temperature and at 37 C (T1/2 ~18 days); however, white variants were significantly more resistant to chloride. Spontaneous switching from white to red was accompanied by loss of chloride resistance. These results positively correlate the white phenotype with chloride resistance. The experiments that led to the conclusions outlined above provided good opportunities to evaluate our new methods. When chloride-treated cells were plated to obtain viable counts, they also were assayed for pre-rRNA content after nutritional shift-up. Samples taken before and after 4-hour chloride treatment were transferred to culture medium and incubated for 3 days to promote RNA synthesis in viable cells. Extracted RNA was then applied to slot-blot membranes and hybridized to a pre-rRNA-targeted probe. Hybridization signals were quantified by phosphoroimage analysis. Pre-rRNA results agreed with those of colony counts, but were available a week faster and with less hands-on effort. However, sensitivity of pre-rRNA measurement was not as good as expected from preliminary data obtained with other bacterial species. To address this problem, we also measured mature rRNA content after nutritional shift-up of chloride-treated and non-treated cells. Contrary to predictions based on the same preliminary data, specificity of mature rRNA probes for viable cells (as opposed to dead cells) was as good as that of pre-rRNA probes. A significant advantage to detecting mature rRNA is its high copy number in M. avium, which allows us to detect it with 10- to 100-fold better sensitivity than pre-rRNA. Although contrary to our initial paradigm, we are examining the possibility that mature rRNA, not pre-rRNA, may be the best target for detecting viable M. avium in drinking water.
Future Activities:
We will continue to evaluate mature rRNA synthesis alongside pre-rRNA synthesis as markers of viable M. avium in drinking water. As part of this effort, we will evaluate the use of commercially available, user-friendly M. avium rRNA detection kits. We will examine the effects of sample decontamination procedures on red and white variants. These procedures commonly are used to destroy competing microorganisms prior to plating drinking water samples for detection of M. avium. We will determine the optimum cultivation conditions for BrdU uptake and incorporation by M. avium. We will initiate efforts targeted to H. pylori. This work will begin in the second half of Year 2 with development of pre-rRNA probes and first-generation BrdU-DNA tests.Journal Articles:
No journal articles submitted with this report: View all 6 publications for this projectSupplemental Keywords:
human health, water, drinking water, infectious disease, decisionmaking, epidemiology, biology, genetics, probes, environmental testing, analytical, measurement, Washington, WA., RFA, Scientific Discipline, Water, Geographic Area, Health Risk Assessment, Environmental Chemistry, State, Environmental Monitoring, Drinking Water, monitoring, microbial contamination, pathogens, Safe Drinking Water, human health effects, microbiological organisms, detection, exposure and effects, disinfection byproducts (DPBs), exposure, mycobacterium avium complex, kinetics, community water system, Washington (WA), genotoxicity, infectious disease, treatment, microbial risk management, water quality, drinking water contaminants, heliocobacter pylori, infectivity, water treatmentProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.